The overall goal of this procedure is to dissociate and culture in vitro, embryonic, sea elegant cells. This is accomplished by first growing large quantities of grave animals. Next eggs are isolated from adults.
Then the eggs are treated with chitinase to dissolve the eggshell. Finally, the cells are manually dissociated and plated for culturing. Ultimately, results can be obtained that show expression of cell specific markers through immunofluorescence, microscopy, electrophysiology, and calcium imaging techniques.
Demonstrating the procedure will be rel ti, a postdoc mal laboratory. After culturing at least four plates of sea elegance to the GR adult stage and preparing media and solutions according to the text protocol. Begin egg isolation by first hawing a tube of previously prepared peanut lectin stock solution.
Place autoclave cover slips in the wells of a 24 well plate and add 200 microliters of peanut lectin to each cover slip incubate for at least one hour at room temperature. Next, aspirate the solution and use sterile water to wash the wells. Once removing all traces of peanut lectin to prevent cell clumping.
Then using sterile autoclave water, wash the GR adults off the agar plates and collect the suspension in two sterile conical 50 milliliter tubes. Place the tubes on ice for up to five minutes to allow the worms to settle to the bottom With a transfer pipette, remove the water and replace it with fresh, sterile autoclave water centrifuge. The worms at 200 G for 10 minutes.
After the final wash, resus, suspend the worms in fresh sterile water and transfer them to a sterile 15 milliliter conical tube before spinning again at 200 G for 10 minutes. If the worms are not completely pelleted, place the tube on ice for five minutes. Once the worms are completely settled, remove the water and add five to six milliliters of lysis solution.
Then gently rock the suspension for five to 10 minutes and every two to three minutes, place a drop of suspension on a cover slip and check for lysis under a stereo microscope. When 72 80%of the worms are lysed, stop the lysis reaction by adding nine milliliters of egg buffer before spinning from this point forward, light a bunsen burner and keep it on to prevent recontamination of the eggs, carefully remove the supernatant and use egg buffer to thoroughly wash the pellet three to four times until the solution is clear. Next, after removing the final supernatant to separate the eggs from the animal carcasses resus, suspend the pellet in two milliliters of sterile egg buffer and add two milliliters of 60%sucrose in egg buffer.
Mix the eggs well in solution before centrifuging for 20 minutes. At 200 G, carefully remove the tubes from the centrifuge. The eggs will be floating at the top of the solution.
Therefore, use a pipetter and sterile one milliliter tips to transfer the eggs into a fresh sterile 15 milliliter conical tube. Use sterile egg buffer to wash the eggs three times while working under a laminar flow hood. To avoid introducing bacterial contamination, resus, suspend the pelleted eggs in one milliliter of two milligrams per milliliter kinase and transfer them to a fresh 15 milliliter conical tube.
Rock the tube for 10 to 30 minutes at room temperature, depending on the freshness of the enzyme when approximately 80%of the eggshells are digested. Centrifuge the eggs at 900 G for three minutes. After carefully removing the supernatant, add three milliliters of L 15 medium, transfer the eggs into a six centimeter diameter plate and begin manual dissociation using a 10 milliliter sterile syringe with an 18 gauge needle.
To monitor the degree of dissociation, place a drop of suspension into a fresh Petri dish and view under a microscope. Continue until approximately 80%of the cells are dissociated. To remove cell lumps, undigested eggs, and hatched larva, gently filter the suspension through a five micron filter and run an additional four to five milliliters of L 15 medium through the filter to recover the cells to culture the cells.
After centrifuging the filtrate at 900 G for three minutes, resus suspend the cells in complete L 15 medium played one milliliter per well and store plates in a humid sealed chamber at 20 degrees Celsius in ambient air. In order to differentiate isolated embryonic elegan cells must adhere to a substrate. The differentiation process begins about two to three hours after plating and continues for about 24 hours.
The morphological features in vitro are remarkably similar to those in vivo. For example, as shown here, a LM and PLM touch neurons develop only two neuronal processes with one longer than the other as they do in vivo. This suggests that at least some of the molecular mechanisms that drive differentiation in elegan cells are cell autonomous and thus can be recapitulated.
In vitro protein markers and post-translational modifications that are cell specific can also be observed in vitro. For example, alpha tubulin is a sated only in touch neurons in sea elgan as shown here in vitro processes of touch neurons stain with an antibody raised against a sated alpha tubulin. And in addition, as seen in this panel, cultured touch neurons express the toxic mutant channel MEC four D, which causes death of touch neurons in vivo indeed.
GFP expressing neurons prepared from a MEC four DPE 4G FP strain are initially present in culture, but then degenerate. However, just as in vivo, the cells can be rescued from death by treatment with a IDE and dantrolene as shown here. Patch clamp techniques are used to study potassium onic, onic, non-selective and dopamine transporter dependent channels in sea elgan cultured cells.
Because of the cell's small size glass pipettes must have a small tip and then a wide cone to offset the increased input resistance. In addition, greater success is achieved by applying a high voltage impulse to the patch of membrane under the tip of the pipette versus gaining whole cell access via suction that can damage the cell. In this figure to study the role of voltage gated calcium channels, ION four is used to perme the membrane and to calibrate touch neurons expressing chameleon YC two point 12 for calcium imaging in the absence or presence of calcium.
In addition, it was determined that the average free calcium concentration in touch neurons is 200 nanomolar molar. After watching this video, you should have a good understanding of how to isolate and culture vitro. See elegance, embryonic cells.
